Paride Mantecca University of Milano Bicocca,

Department of Earth and Environmental Sciences

Research Centre POLARIS,

Milan, Italy

Biological Effects of Nanoparticles

“Nanoparticles in the environment: fate and effects”,

Tallin, 29 May 2014

The story of the University of Milano Bicocca

Pirelli industrial site

Campus Bicocca

http://www.polaris.unimib.it/index.php/en/who-we-are/nanotoxicology

www.polaris.unimib.it

RESEARCHES

The story of the POLARIS Research Centre (Dept. Earth and Environmental Sciences, University of Milano Bicocca)

From tire particles analysis

…and toxicity

To Nanoparticle biological

effects and MOA

Through Particulate Matter

health effects

Milan

The inhalable fraction of Tire Particles induce (lung) toxicity

… and the effects are related to size

The fine fraction resulted enriched with sub-micrometric

(nanometric) particles

In London, in 1952, 3-4.000 people died as a

consequence of 3 days of great smog of PM and

sulphur dioxide.

London’s Great Smog

Statistical average loss of life expectancy in months in Europe

due to anthropogenic PM 2.5 (air pollution)

The spectre

PM is a relevant environmental and health concern

Stimulus also for the scientific community:

PM effects may be related not only to mass…

Press and

public opinion

asked for

evidences…

TOSCA project: Toxicity of Particulate Matter and molecular Markers of Risk

Urban PM10 and PM2.5 sampled in

Milan in summer and winter

PM10

PM2.5

Teflon filter

BEAS-2B

Exposure of in vitro

systems representative

of the lung epithelia

A549

THP-1

In vivo

toxicology

Clinical and epidemiological

studies

Bacteria Virus

Natural and anthropogenic particles

Cigarette smoke

Allergens

PM is a complex mixture of particles (μm and nm scale)

from different sources

Oberdorster et al., 2005 (EHP)

Penetration of particles in the respiratory system

Per cent distribution of emissions in Lombardy for 2010 by fuel

Fuel / Pollutnat PM2.5 PM10 TSP

gasol ine 0,52% 0,45% 0,39%

coal 0,72% 0,73% 0,99%

diesel 19,65% 17,02% 14,51%

refinery gas 0,50% 0,43% 0,37%

gasoi l 0,58% 0,50% 0,42%

LPG 0,04% 0,03% 0,03%

kerosene 0,09% 0,09% 0,08%

wood 56,14% 49,51% 44,66%

natura l gas 1,01% 0,94% 0,87%

fuel oi l 1,26% 1,13% 1,01%

others 1,75% 1,74% 1,85%

no fuel 17,73% 27,42% 34,83%

Total 100,00% 100,00% 100,00%

Combustion processes – fine and ultrafine (nano) particles

Buzea et al., 2007

31.3

73.4

16.8

52.2

10.4

32.3

4.3 6.50

0

10

20

30

40

50

60

70

80

90

100

ESTATE INVERNO

mg

m-3

Concentrazioni

PM10

PM2.5

PM1

PM0.4

18% 15%

20% 34%

16%

28%

39%23%

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

ESTATE INVERNO

Distribuzione dimensionale

PM(10-2.5) PM(2.5-1) PM1 PM0.4

31.3

73.4

16.8

52.2

10.4

32.3

4.3 6.50

0

10

20

30

40

50

60

70

80

90

100

ESTATE INVERNO

mg

m-3

Concentrazioni

PM10

PM2.5

PM1

PM0.4

18% 15%

20% 34%

16%

28%

39%23%

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

ESTATE INVERNO

Distribuzione dimensionale

PM(10-2.5) PM(2.5-1) PM1 PM0.4

Fine and Ultrafine Particles abundant in urban PM

Prog TOSCA, 2011

… also in Milan, especially in Winter

Background

Summer PM10

SO4=

15,4

NO3-

5,4

NH4+

6,4

EC

10,1

Metals

4,8

Inorganic

ions

4,8

OC

33,0

unaccounted

20,0

Winter PM10

unaccounted

28,2

OC

33,0

Inorganic

ions

5,4

Metals

2,5

EC

8,2

NH4+

5,4

NO3-

13,5

SO4=

4,3

Bacterial components (endotoxin content)

Chemical characterization (mass %)

Summer PM10 effect

Pro-inflammatory effect

release of the pro-inflammatory

mediator IL-1β

Release of IL-1β triggered by Milan summer PM10: molecular pathways involved in the cytokine release. R

Bengalli, E Molteni, E Longhin, M Refsnes, M Camatini, M Gualtieri. http://dx.doi.org/10.1155/2013/158093

Low doses

(5 and 2.5 μg/cm2)

Summer PM10 characterization

Background

Summer PM2.5

unaccounted

14,8

OC

28,8

Inorganic

ions

2,0Metals

2,3

EC

14,6

NH4+

11,6

NO3-

3,6

SO4=

22,4

Winter PM2.5

SO4=

6,4

NO3-

20,6

NH4+

9,1

EC

11,8

Metals

0,9

Inorganic

ions

3,2

OC

35,9

unaccounted

12,1

Chemical characterization (mass %) PAHs (OC %)

OC

%

Winter PM2.5 characterization

Winter PM2.5 effect

0

10

20

30

40

50

60

70

SubG1 G0/G1 S G2/M

Cel

ls %

Ctrl Summer PM2.5 Winter PM2.5

0

2

4

6

Control summer winter

PM2.5

Flu

ore

scen

ce a

.u.

0

2

4

6

Control summer winter

PM2.5

flu

ore

scen

ce a

.u.

ROS formation DNA damage γH2AX expression

Cell cycle alteration

PM dose: 10 μg/cm2 *p<0.05 vs Ctrl

Increased mitotic cells

* * *

*

0

10

20

30

40

50

60

70

80

90

subG1 G0/G1 S G2/M

Ce

ll n

um

be

r (%

)

*

*

0

10

20

30

40

50

60

70

80

90

subG1 G0/G1 S G2/M

Cell

num

ber

(%)

Control whole washed organic

0

10

20

30

Control whole washed organic

PM2.5

Flu

ore

sce

nce

(a

.u.)

*

*

ROS formation Cell cycle alteration

* p<0.05 vs Ctrl

Whole FP Acetonitrile,

centrifuge (15min, 13000rpm)

Organic fraction

resuspended in

DMSO

Washed FP

resuspended in

H2O

How we obtained

the organic

fraction Carbon cores

Adsorbed hydrocarbons

The organic soluble fraction of PM2.5 is responsible for the

genotoxic effects. Washed particles (BC?) are ineffective

Toxicity of UFP from Diesel with different additives

Carbon

cores

Adsorbed

hydrocarbons

Diesel emissions:

NOC vapour

phase

hydrocarbo

ns

Cell viability

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Diesel Diesel + alkyl benzene Diesel + naphteneF

old

In

cre

ase

1ppm 3ppm 7ppm

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Diesel Diesel + Alkyl

benzene

Diesel +

naphtene

Biodiesel

Fo

ld In

cre

ase

ROS formation

0

1

2

3

4

5

6

7

Ctrl Diesel Diesel + alkyl

benzene

Diesel + naphtene

RO

S (

a.u.)

*

*

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Diesel Diesel + alkyl benzene Diesel + naphtene

Fo

ld In

cre

ase

1ppm 3ppm 7ppm

Winter FP organic

compounds /

PAHs

DNA damage CELL

NUCLEUS

Cell cycle

alteration

ROS

3. Mitotic spindle involvement

in cell cycle alteration

2. ROS/CYP

induced DNA

damage

1. AhR/CYP

activation

Cell death and

carcinogenesis

AhR

AhR

Possible mechanism of action of fine PM (and combustion derived UFPs)

The PM toxicity depends not only from the concentration

Biological effects vary according to the particle size and season of collection

PM chemical composition (and thus the contribution of different emission

sources) stands at the base of the cell response variability

Coarse PM-induced responses are much more related to acute inflammatory

events

Fine and ultrafine fractions mainly originate from combustion sources and

their biological activity is mainly related to pro-carcinogenic events

Data on health effects of UFPs from different combustion sources are

incosistent. There’s the need to develop new toxicity paradigm and

technology

Summary of the PM and UFP biological effects

Exposures to airborne nanosized particles (NSPs; < 100

nm) have been experienced by humans throughout their

evolutionary stages, but it is only with the advent of the

industrial revolution that such exposures have increased

dramatically because of anthropogenic sources such as

internal combustion engines, power plants, and many other

sources of thermodegradation.

The rapidly developing field of nanotechnology is likely to

become yet another source for human exposures to

NSPs—engineered nanoparticles (NPs)—by different

routes: inhalation (respiratory tract), ingestion

[gastrointestinal (GI) tract], dermal (skin), and injection

(blood circulation).

Nanotoxicology was born (term coined in 2004-2005)

When peculiar phisical chemical properties of NPs

suggested unpredictable interactions with living systems

Evolution of “traditional” particle and fibre toxicology

Donaldson et al., 2004 – Occup. Environ. Med. 61:727-28

Oberdorster et al., 2005 – Environ. Health Perspect. 113:823-39

Seaton and Donaldson, 2005 – Lancet 365:923-24

From coal minings to nanotechnology

… and the advent of nanotoxicology

Biological interactions and effects of Engineered Nanoparticles (ENPs)

NP properties and biological interactions

Nanomaterials properties driving

adverse effects

Correlations between phisico-chemical properties

of NMs and biological and toxicological outcomes

(Schvedova et al., 2010)

•Dose-dependent toxicity

not always NP effects are correlated with particle

mass dose (high conc, high agglomeration?!)

•Size-dependent toxicity

•Surface-area-dependent toxicity

•Crystalline-structure-dependent toxicity

•Surface-coating dependent toxicity

Figure 4. Percentage of neutrophils in lung lavage of rats (A,B) and mice (C,D) as

indicators of inflammation 24 hr after intratracheal instillation of different mass doses

of 20-nm and 250-nm TiO2 particles in rats and mice. (A,C) The steeper dose

response of nanosized TiO2 is obvious when the dose is expressed as mass. (B,D)

The same dose response relationship as in (A,C) but with dose expressed as particle

surface area; this indicates that particle surface area seems to be a more appropriate

dosemetric for comparing effects of different-sized particles, provided they are of the

same chemical structure (anatase TiO2 in this case). Data show mean ± SD.

Oberdorster et al., 2005 (EHP)

NP properties relevant for toxicological studies

Griffitt et al., 2007. Environ. Sci. Technol. 41:8178-8186

Looking for sensible markers able to discriminate the effects of NM

Toward a biology of nanosystems

… where nanotoxicology is defined

as the study of the interference of

man-made nanomaterials with

endogenous (cellular)

nanostructures.

(Shvedova et al., 2010)

“Nanovision”

Living cells as a miniature factory that

contain a large collection of dedicated

protein machines of nano-scale

dimensions, optimized by billions of years

of evolution.

Alberts B., 1998. The cells as a collection of

protein machines: preparing the next generation

of molecular biologists. Cell 92:291-94

The evolution of nanotoxicolgy into a predictive science as

apposed to being merely descriptive science

Nanostructural Biology: a new frontier for nanotoxicology?

Nanotoxicology Unit Ref. Paride Mantecca (Dept. Earth and Environmental Sciences, University of Milano Bicocca)

In vitro nanotoxicology M. Gualtieri, C. Urani, E. Longhin, R. Bengalli,

L. Capasso, M. Camatini

Cell models available:

- Human lung cells (A549, NCI-H441;

BEAS-2B) ;

-human monocytes (THP-1)

-Human lung microvascular endothelial

cells (HPMEC-ST1.6R)

- mouse fibroblasts (C3H) – to study

general cytotoxicity and cytoskeletal

remodelling

Applications:

- NP-induced cytotoxic effects

- Production of inflammatory mediators

- NP-induced genotoxicity

- NP properties driving cytotoxicity

(size, shape, ion dissolution, surface

reactivity)

Aquatic nanotoxicology A. Colombo, P. Bonfanti, E. Moschini

Collaborators: R. Bacchetta, P. Tremolada, L.

Del Giacco (University of Milan)

Aquatic models available:

- Frog Embryo Teratogenesis Assay –

Xenopus, FETAX – (amphibian

developmental toxicity test, ASTM

E1439-98)

- Daphnia magna acute and chronic

toxicity test (ISO 6341, 10706)

Applications:

- Acute, chronic and developmental

toxicity of nude and functionalized NPs;

NPs and soluble contaminants leached

from nanostructured materials; effluents

and contaminated surface waters.

- Biological mechanisms inducing NP

toxicity in aquatic organisms

Microscopy and Spectroscopy of

cell-NP interactions G. Chirico, M. Collini, L. D’Alfonso, S. Freddi

Collaborators: U. Fascio, N. Santo (CIMA,

University of Milan)

Techniques

- Conventional and analytical light and

electron microscopy

- Confocal laser microscopy

- Two Photon Correlation Spectroscopy

- Stimulated emission depletion (STED)

microscopy (nanoscopy)

-Dynamic Light Scattering

Applications:

-Modality of cell-particle interaction

-- NP tracking and translocation in fixed

and living systems

- Structural characterization of NP

suspensions.

1- Toxicity and mode of action of metal oxide NPs in human lung cells

MeO NPs (CuO in particular) are highly toxic to lung cells

Cytotoxicity is driven by abundant NP internalization and a Trojan horse-mediated

autophagic cell death

2- Toxicity and mode of action of metal oxide NPs in embryos

Comparative embryotoxicity: the teratogenic potential of ZnO NPs and the dissolved ions-

mediated effects of nCuO

Permeation and disruption of the intestinal barrier: a specific MOA for nZnO?

3- Toward the reduction of NP toxicity: surface coating and crystallinity

modulation

Does nZnO capping by polymers help in reducing toxicity?

Lower the crystallinity, lower the toxicity of CuO NPs

4- Metal oxide nanocomposite showing enhanced reactivity… and

toxicity?

… more effective than CuO and ZnO, less toxic than Zn

Biological effects of ENPs: the case study of nano metal oxide

**

**

*

* *

0

20

40

60

80

100

120

0 1 5 10 50 100

nCuO (ug/ml)

24h

-Via

bil

ity%

(M

TT

)

nCuO Cu++ Dissolved Cu++

- CuO NPs are highly cytotoxic

- Toxicity depends on NP

internalization and reactivity

The modality of cell-NP interaction drives nCuO toxicity in A549 cells

Moschini et al. 2013. Toxicology Letters 222: 102– 116

nCuO

nTiO2

1- Toxicity and MOA of metal oxide NPs in human lung cells

Lysosome

Late

En

do

so

me

Lyso

so

mes

Moschini et al. 2013.

Toxicology Letters 222: 102– 116

Trojan horse mechanism and autophagic cell death in nCuO exposed cells

- CuO NPs endocytosis and

lysosomal destabilization

- NP intracellular dissolution

and autophagy induction

1- Toxicity and MOA of metal oxide NPs in human lung cells

- No mortality

- Significant malformation rates

- Significant growth inhibition

Co CuO

Co ZnO

2- Toxicity and MOA of metal oxide NPs in embryos

Comparative embryotoxicity of Metal oxide NPs (nCuO, nZnO, nTiO2)

ZnO NPs are potential teratogen.

Malformations specifically affect

intestine

Bacchetta et al., 2012. Nanotoxicology 6 (4): 381-398

0

20

40

60

80

100

Co

CuO

100

CuO

500

Cu+

+

ZnO

100

ZnO

500

Zn+

+

malf

orm

ed

%

* #

*

* *

C

o CuO Cu++

ZnO

Zn++

CuO NPs effects are mediated by

dissolved ions

F-actin

ZO-1

2- Toxicity and MOA of metal oxide NPs in embryos

ZnO<50 ZnO<100

nZnO MOA: permeation and disruption of the intestinal barrier

bZnO Co sZnO

TBARS assay

RT-PCR

4-HNE immunofluorescence

Bacchetta et al., 2013. Nanotoxicology, Aug, early on line

1- NPs penetrate apical membrane 2- … walk along the lateral membrane

and paracellular space…

3- … reach basal membrane and

underneath connective tissue

4- … induce swelling and finally

cell death

- Nano-sized ZnO is absorbed by epithelial cells and

damages intestinal mucosa reaching underlying tissues

- The mechanism involves oxidative stress, disruption of

cytoskeleton and cell junctions, independently from

dissolved Zn++.

- The loss of cell junction integrity facilitates the NP

translocation and leads to cell death.

Bacchetta et al., 2013. Nanotoxicology

2- Toxicity and MOA of metal oxide NPs in embryos

0

20

40

60

80

100

Control SZnO 50 BZnO 50 SZnO 50 BZnO 50 SZnO 50 BZnO 50

Nude PVP PEG

Experimental groups

ma

lfo

rme

d e

mb

ryo

s (

%)

?

Small/round Big/rod

*

*

*

**

0

20

40

60

80

100

Control SZnO

[1]

SZnO

[10]

SZnO

[50]

SZnO

[100]

BZnO

[1]

BZnO

[10]

BZnO

[50]

BZnO

[100]

Experimental groups

%

Mortality (%) Malformed larvae (%)

? ?

Differently shaped nZnOs displayed comparable effects (?)

Modification of ZnO NPs size and surface coating

Polymer-capped ZnO NPs produced effects comparable to

those of nude ones (?)

Lower the crystallinity, lower the toxicity of CuO NPs

CuO and ZnO NPs sonochemically synthesized (Bar-Ilan Univ)

crystallites with more defects and less organized structure are

more toxic to the bacteria…

… and sono-CuO NPs are less toxic to Xenopus embryos and

human lung cells…

Toward the reduction of NP toxicity

-1

0

1

2

3

4

5

6

7

8

9

10

CuO - com CuO - sono

An

tib

acte

rial

act

ivit

y va

lue

(A

)

0

1

2

3

4

5

6

7

ZnO - com ZnO - sono

Ant

ibac

teri

al a

ctiv

ity

valu

e (A

)

CuO

ZnO

Commercial

Sono

DSC Antimicrobial activity

Sono

Sono

(Perelshtein et al. Nano Research, in press)

Striped bars= Sono Solid bars=commercial

A metal oxide nanocomposite with enhanced antibacterial properties

… and toxicity?

CuO

ZnO

Cu0.89Zn0.11O

b

Zn-doped CuO nanocomposite (Zn-CuO)

Eradication of Multi-Drug

Resistant Bacteria by a Zn-

doped CuO Nanocomposite

Malka et al. 2013. Small (in

press)

S. aureus E.coli

MRSA MDR E.coli

*

*

*

0

20

40

60

80

100

Contr

ol

0,1 1

10

100

0,1 1

10

100

0,1 1

10

100

CuO ZnO Zn-CuO

Malf

orm

ed

larv

ae (

%)

Experimental group

0

20

40

60

80

100

120

con

trol 1

10

100 1

10

100 1

10

100

CuO MAE ZnO MAE Zn-CuO

Via

bil

ity%

NP (ug/ml)

3h 6h 24hOn amphibian development On cell viability

Need for advanced in vitro models for assessing NPs toxicity

NPs-induced cardio-vascular effects likely involve ABB alterations. NPs may exert its effects through the ABB translocation or by inflammatory mediators released by the epithelium.

NPs

1. Local effects:

- inflammation

- oxidative stress

- DNA damage

2. Systemic effect:

cardio-

vascular

diseases

Possible mechanisms of PM

cardio-vascular effects:

• ABB translocation

• Release of inflammatory mediators by

epithelium and endothelium

A model of air-blood barrier

In vitro reconstructed Air-Blood Barrier (ABB): 3D co-cultures

NCI-H441

HPMEC-ST1.6R

THP-1

Lung epithelium

Lung endothelium

Monocytes

What is still almost unknown in UFP and NP toxicology

Carcinogenic and neurodegenerative effects

Paracelsus Philippus Aureolus Theophrastus

Bombastus von Hohenheim (1493 – 1541)

Funder of the discipline of toxicology.

« Omnia venenum sunt: nec sine veneno

quicquam existit. Dosis sola facit, ut venenum

non fit »

«"All substances are poisons; there is none

which is not a poison. The right dose

differentiates a poison…."

The dose makes the poison…

… for toxicologists since 1500… what’s now for NPs?

Concluding remarks

- Different incidental and engineered NPs show different mode of action in different biological

models

- Toxicity levels vary according with NP properties, like size, extra- or intra-cellular solubility,

crystallinity and chemicals associated (or adsorbed onto) primary particles

- The knowledge of the modality of cell-NP interactions and the molecular pathways driving

the biological effects are crucial in assessing NP toxicity

-The development and use of more complex in vitro systems are necessary to predict the

complex biological responses to NPs in vivo

- Only addressing the complexity of the biological responses to NPs it is possible to generate

toxicological data useful in risk assessment. At present many gaps have to be filled in NP

exposure monitoring and toxicity during the NP entire life cycle (especially for ENPs)

- Special attention should be devoted to the effects after prolonged exposure periods to low

NP doses (in mass), but high in number.

- A strong multidisciplinary approach is the only possible way toward succeeding in limiting

environmental and human health consequences to NP exposure.

Current projects in which we are trying to address these issues - Biological effects and human health impacts of ultrafine particles sources (new generation diesel and biomasses)

- Do new generations of nano-antibacterials OVERcome the epithelial barriers posing human health at risk? A predictive

nanoTOXicology study (OVER NanoTOX)

- The transfer of engineered nanomaterials from wastewater treatment & stormwater to rivers (ENTER) EU-COST Action ES1205

- Mode of action of engineered nanoparticles (MODENA). EU-COST Action

(DISAT)

Marina Camatini

Maurizio Gualtieri

Eleonora Longhin

Elisa Moschini

Laura Capasso

Rossella Bengalli

(DSS)

Paola Palestini

Giulio Sancini

Alberto Pesci

Francesca

Farina

Particle Toxicity Group

Paride Mantecca, Università degli Studi di Milano Bicocca, Dipartimento di Scienze dell’Ambiente e del Territorio e di Scienze

della Terra, 1, piazza della Scienza, 20126 – Milano. Tel. 02 6448 2916, e.mail: paride.mantecca@unimib.it